Project Summary Lynch syndrome (LS), the most common hereditary cancer condition, is usually caused by a heritable genetic mutation affecting one of the DNA mismatch repair genes, most frequently MLH1, located on chromosome 3. So-called ?MLH1 epimutation? is an alternative cause for LS. MLH1 epimutations are characterized by methylation of a single copy of the MLH1 promoter, accompanied by loss of gene activity, throughout normal tissues. This predisposes carriers to the development of LS-type cancers. Patients are identified by the detection of ?constitutional? MLH1 methylation in DNA from blood or any other normal tissue. Studies of the family members of cases with an MLH1 epimutation have revealed distinct patterns of inheritance and hence differing risks of passing the epimutation from parent to offspring, if at all. Therefore, MLH1 epimutations provide a unique opportunity in humans for studying how epigenetic alterations arise and thereby cause disease, and also how epigenetic alterations are transmitted from one generation to the next in some families, but not in others. Dr. Hitchins, the principle investigator of this study, has assembled a unique collection of families with an MLH1 epimutation as the cause for cancer, and has established the inheritance patterns of the epimutation in each family, through her work in this field over the past 13 years. The goals of this study are to determine the mechanisms that underlie MLH1 epimutation by undertaking an in-depth study of the genomes and epigenomes of the patients and their families. In Aim 1, we will use state of the art ?linked-read whole genome sequencing? to sequence the entire genome of selected patients and family members to determine if various types of genetic mutations on chromosome 3, or other chromosomes, underlie the onset on MLH1 epimutations. In Aim 2, we will determine if MLH1 is the only gene that is subject to erroneous constitutional methylation, or if other genes are simultaneously methylated. This would provide insights into whether epimutations arise because of a generalized epigenetic perturbation, or a focal mechanism affecting MLH1 alone. In Aim 3, we will validate any genetic or epigenetic alterations identified and determine their segregation patterns in families. We will also perform functional analyses of genetic variants identified previously and in Aim 1. Collectively, these studies will provide evidence for a causal link between genetic alterations identified and the MLH1 epimutation, and enable us to predict the likelihood of inheritance between generations. Finally, we will study patient white blood cells induced into a pluripotent state to model the epigenetic reprogramming events that occur at MLH1 in these patients. This will shed light on why some epimutations are inherited and others not. If successful, this study will enable us to improve the clinical management and genetic counselling of families with a cancer-predisposing MLH1 epimutation. More broadly, this study will provide significant new insights into the interaction between disease-causing genetic and epigenetic states, as well how altered epigenetic states may are passed from one generation to the next, or erased between generations.